Molecular Recognition by Clathrin Adaptors[unreadable] [unreadable] The clathrin coat plays a ubiquitous and fundamental role in endocytosis and in endosomal sorting within the eukaryotic cell. Clathrin forms a cage that surrounds cargo-bearing vesicles, but clathrin itself does not directly bind to cargo. Cargo is sorted into clathrin-coated vesicles by adaptor proteins that physically bridge cargo and clathrin. The best-known general purpose adaptors are the heterotetrameric adaptor protein complexes (AP complexes) and the multimodular GGA adaptor proteins. The goals of this project are 1) to identify the binding sites for cargo on adaptor proteins and measure their affinities quantitatively; 2) to determine the crystal structures of complexes between adaptors and soluble cargo fragments; and 3) to relate structure and function using mutational analysis.[unreadable] [unreadable] Specific sorting signals direct transmembrane proteins to the compartments of the endosomal-lysosomal system. The tyrosine-based sorting signal binds to the ear domains of the large subunits of AP complexes (AP-1, -2, -3, and 4), and has been well characterized. Acidic-cluster-dileucine signals of the form DXXLL present within the cytoplasmic tails of sorting receptors, such as the cation-independent and cation-dependent mannose 6-phosphate receptors, are recognized by the VHS domain of the GGA adaptor proteins. In previous work, our laboratory determined the structure of the GGA VHS domain bound to DXXLL-motif peptides. We went on to characterize all of the domains of the GGA proteins in complex with relevant protein or peptide ligands. The DXXXLL motif exists in medically important host proteins such as CD4 and viral proteins such as HIV-1 Nef. Unlike the DXXLL motif, it binds to AP complexes, not GGAs. Despite its importance and substantial history, and years of effort by multiple laboratories, the structural basis for the DXXXLL motif is unknown. To address this problem, our lab generated a variant AP-2 complex that is competent to bind to DXXXLL motif peptides.[unreadable] [unreadable] CD4 downregulation depends on a conserved (D/E)XXXL(L/I)-type dileucine motif in the C-terminal, flexible loop of Nef, which mediates binding to the clathrin adaptor complexes AP-1, AP-2, and AP-3. In 2007-8, we collaborated with the Bonifacino lab (NICHD) to identify a consensus (D/E)D motif within this loop as a second, conserved determinant of interaction of Nef with AP-2, though not with AP-1 and AP-3. The principal role of our group was to purify the variant form of the AP-2 core competent to bind to Nef. Mutations in this diacidic motif abrogate both AP-2 binding and CD4 downregulation. A dileucine motif from tyrosinase, both in its native context and in the context of Nef, can bind to AP-2 independently of the diacidic motif. These results identified a novel type of AP-2 interaction determinant, supporting the notion that AP-2 is the key clathrin adaptor for the downregulation of CD4 by Nef, and revealed a previously unrecognized diversity among dileucine sorting signals.